Rapid Communication J Biochem.135, 285-288 (2004) DOI:10.1093/jb/mvh034 DNA Display of Biologically Active Proteins forIn Vitro Protein Selection Masato Yonezawal,2, Nobuhide Doi1, Toru Higashinakagawa2 and Hiroshi Yanagawa*,1 1 Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522; 2Department of Biology, School of Education, Waseda University, 1-6-1 Nishi-waseda, Shinjuku-ku, Tokyo 169 8050 Received December 20, 2003; accepted January 9, 2004 In vitro display technologies are powerful tools for screening peptides with desired functions. We previously proposed a DNA display system in which streptavidin-fused peptides are linked with their encoding DNAs via biotin labels in emulsion compart ments and successfully applied it to the screening of random peptide libraries. Here we describe its application to functional and folded proteins. By introducing peptide linkers between streptavidin and fused proteins, we achieved highly efficient (>95%) formation of DNA-protein conjugates. Furthermore, we successfully enriched a glu tathione-S-transferase gene by a factor of 20-30-fold per round on glutathione-cou pled beads. Thus, DNA display should be useful for rapidly screening or evolving pro teins based on affinity selection. Key words: biotin, DNA display, in vitro translation, protein engineering, streptavidin. Abbreviations: aa, amino acid; SA, streptavidin; GST, glutathione-S-transferase; BLIP, ƒÀ-lactamase inhibitory protein; RYBP, Ringl and YY1 binding protein. Determining the functions of proteins is an important assembly of virion particles are not displayed. Indeed, task in the process of applying data from the human only proteins containing less than 200 amino acids (aa) genome project to achieve new advances in therapeutics, have been screened from phage-displayed cDNA libraries diagnosis, and medicine. One approach is to search for (8-11). Previously, we developed an in vitro DNA display interaction partners, including ones for protein-protein, system called 'STABLE' (12), which allows the complete small molecule-protein, and nucleic acid-protein interac in vitro construction of DNA-displayed peptide libraries. tions. So far, several methods have been used. The yeast Recently, we showed that short linear peptides that bind two-hybrid approach has been a leading technology for to a monoclonal antibody could be screened from a ran identifying protein interactions (reviewed in Ref. 1). dom peptide library (13). In the present study, we have However, it has significant limitations that arise because displayed a folded, functional protein on linear DNA and the interactions take place in a living system. Mass spec selectively enriched a glutathione-S-transferase (GST) trometric identification of proteins after affinity chroma gene by affinity purification with glutathione-immobi tography is also a powerful technology (reviewed in Ref. lized beads. 2). However, this approach often fails after laborious and In vitro compartmentalization utilizing water-in-oil time-consuming efforts to afford sufficient amounts of emulsions was originally developed by Tawfik and Grif purified proteins due to low-level expression or inade fiths to cage a single gene per micelle and to select cata quate purification. Protein microarrays (Ref. 3 and refer lytically active proteins (14). In our DNA display strategy ences therein) are another alternative for systematically (Fig. 1), proteins are expressed as streptavidin (SA) identifying protein-protein interactions and drug recep fusion proteins in order to conjugate the proteins to their tors. However, they require the preparation of a large encoding DNAs with biotin labels in emulsion compart number of proteins and the use of special equipment such ments. The resulting DNA-protein conjugates are as microarrayers. selected by affinity purification followed by PCR amplifi Display technologies that link genotype and phenotype cation. molecules comprise a powerful alternative [reviewed in Since the conjugation of a protein to its encoding DNA (4-6)]. Large libraries can be screened iteratively by is a crucial step in DNA display, we examined whether or amplifying selected genes in host cells or PCR, so even not folded proteins fused to SA can be efficiently conju very low copy number proteins can be identified. Phage gated to a biotinylated DNA (Fig. 2). When a BLIP gene display (7) is the most widely used display technology, (encoding a 165-aa protein, Ref. 15) was directly fused to though its use has been significantly hampered by the the SA gene and expressed as a fusion protein, about half limitations of producing libraries in a living system. As of the input DNA was not conjugated to the protein (Fig. proteins are expressed as fusions to phage coat proteins 2B, lanes 1 and 2). To reduce steric hindrance , which may in bacterial cells, those that are toxic or that abrogate the affect the formation of DNA-protein conjugates, peptide linkers were introduced between SA and BLIP (Fig. 2A). We chose a -22-aa linker containing a hemagglutinin-tag *To whom cor respondence should be addressed. Tel: +81-45-566 (HA), a -50-aa linker containing a hemagglutinin-tag 1775, Fax: +81-45-566-1440, E-mail: [email protected] and a glycine/serine-rich sequence (GS), which is often Vol. 135, No. 3, 2004 285 ? 2004 The Japanese Biochemical Society. 286 M. Yonezawa et al. Fig. 1. A schematic representation of the DNA display selec tion procedure. (1) A DNA library encoding SA-fusion proteins is labeled with biotin and compartmentalized in a water-in-oil emul sion containing an in vitro transcription/translation system. (2) In each compartment, SA-fusion proteins are synthesized and attached to the template DNA via biotin labels. (3) DNA-protein Fig. 2. Formation of DNA-protein conjugates. (A) A schematic conjugates are recovered from the emulsion and (4) subjected to representation of DNA constructs for in vitro transcription/transla affinity selection on an immobilized bait. (5) After washing and elu tion. DNA constructs were labeled by PCR with fluorescein at the tion, the DNA portion of the bound molecules is amplified by PCR. upstream ends and with biotin at the downstream ends using (6) DNA is subjected to the next round of selection or (7) identified labeled primers as described (13). Plasmids were constructed by by sequencing. Although SA-fusion proteins form a tetramer and modifying pSta4 (13) using standard subcloning techniques (28). thus four copies of protein can be displayed, only one copy of the The translated open reading frame consists of sequences for a T7 protein is shown in this figure for simplicity. tag, streptavidin (SA), a peptide linker, and a fused protein (gene). The amino acid sequences of the linker portions are YPGSQLYPY DVPDYASLGGHMA (22 residues, termed HA), YPGS(GGG GS)5GGGRSQLYPYDVPDYASLGGHMA (52 aa, termed GS) or used in ribosome display, and a -40-aa linker containing SA(EAAAK)4ARSQLENLYFQGGS (36 residues, termed HL). DNA EAAAK repeats (HL), which forms a helical structure fragments coding for BLIP and Rae28/Mph1 were amplified by PCR and effectively separates the domains of bifunctional pro from plasmids carrying these genes (29, 30). GST and luciferase teins (16). The insertion of peptide linkers improved the genes were amplified from pGEX-4T-3 (Amersham) and TNT con trol template (Promega), respectively. Histone H4 and RYBP genes efficiency of formation of DNA-protein conjugates. The were amplified from a mouse testis cDNA library (Clontech). All efficiency reached -70% when HA was introduced (Fig. DNA sequences were confirmed with a CEQ2000 sequencer (Beck 2B, lanes 3 and 4) and >95% when GS or HL was intro man Coulter). (B) In vitro transcription/translation was performed duced (Fig. 2B, lanes 5-8). using a TNT-SP6 coupled wheat germ extract (Promega) with 10 While the total yields of synthesized proteins were nM DNA templates in the presence (+) or absence (-) of 0.1mM free biotin competitor. The reaction products (3 µl) were separated in 2% almost constant, the proportion of tetrameric protein rel agarose gels containing 0.1% SDS prepared with Seakem Gold aga ative to total protein increased when peptide linkers rose (BioWhittaker Molecular Applications). DNA (open arrow were introduced (data not shown). This effect was largely heads) and DNA-protein conjugates were detected via fluorescence dependent on the length of the linkers. These results sug using Molecular Imager FX (Bio-Rad). DNA-protein conjugates ex gest that tetramerization of SA-fusion proteins, which is hibited lower mobility than free DNA. The multiple bands of DNA required for tight association with biotin (17), was - protein conjugates originate from binding of different numbers of enhanced when relatively long peptide linkers were DNA molecules to a tetramerized SA-fusion protein in non-emulsi fied reactions. The size of each displayed protein excluding the SA introduced and that it allowed highly efficient formation and linker portions is indicated in parenthesis. of DNA-protein conjugates. Furthermore, proteins other than BLIP (-20 kinds) whose sizes were in the range of 102-1012 as were fused Next, we addressed whether or not the GST gene can to SA through GS or HL. We also examined C-terminal as be enriched with our DNA display strategy. DNA tem well as N-terminal SA fusions. All proteins showed the plates for SA-fusion of GST or RYBP, as a negative con formation of DNA-protein conjugates with an efficiency trol, were mixed in a ratio of 1:10 or 1:1,000, and then of >95% (Fig. 2B, lanes 9-18, and unpublished data). used for affinity selection on glutathione-coupled beads. It is unclear how large proteins can be used for screen As shown in Fig. 3A, the DNA band corresponding to ing with other in vitro display technologies based on cell GST was enriched after selection. By conducting two free translation, such as ribosome display (18-20) and rounds of selection from the 1:1,000 mixture, GST was mRNA display (21-23).